Significance and context

The Gram-positive eubacterium Streptomyces coelicolor A3(2) is considered a model soil bacterium, with a complex life cycle involving mycelium
growth and spore formation. An interesting feature of S. coelicolor is its capacity to produce useful pharmaceuticals - anti-tumor agents, immunosuppressants
and a variety of antibiotics, including the tripyrrole undecylprodigiosin (Red), calcium-dependent
lipopeptide (CDA) and the polyketide actinorhodin (Act). These compounds are usually
produced during the morphological transition that initiates spore formation. Genes
involved in antibiotic synthesis are mostly clustered on the S. coelicolor genome, and antibiotic synthesis involves a reasonably large number of genes, the
expression of which is controlled by complex regulatory mechanisms. The availability
of the S. coelicolor A3(2) genome sequence and a set of mutants enabled Huang et al. to carry out a global DNA-microarray-based analysis of gene expression during growth
and antibiotic synthesis.

Key results

Growth of S. coelicolor cultures is typically characterized by four stages (see Figure 1): an initial rapid
growth phase, starting approximately 14 hours after inoculation; a transition phase
of approximately 2 to 3 hours, starting 18.5 hours after inoculation; a second rapid
growth phase lasting for approximately 5 hours; followed by a stationary phase. The
production of the antibiotics CDA and Red starts during the transition phase, whereas
Act production is only apparent during the stationary phase. RNAisolated at different times during growth (see asterisks in Figure 1) was hybridized
with 4,960 known genes or putative open reading frames (ORFs) on microarray glass
slides. Almost 25% of the genes tested showed a two-fold or greater change in expression
between at least two time points during growth. Most known genes involved in antibiotic
synthesis were upregulated during morphological transition. Expression of the red and cda genes, involved in the synthesis of Red and CDA, respectively, was upregulated as
soon as these antibiotics appeared in the medium. Interestingly, redZ, a putative initiator of the Red synthetic pathway, was expressed slightly earlier.
Similarly, the act genes, responsible for the synthesis of Act, were only expressed during the stationary
phase, when Act is produced. Comparison of expression patterns of unknown genes with
those of known genes suggested the involvement of additional genes in, for example,
Red synthesis. Gene expression was also studied in S. coelicolor mutants of redD and/or actII-ORF4, genes involved in the synthesis of Red and Act, respectively, in order to discover
other genes whose expression could be regulated by the presence of redD and/or actII-ORF4. The expression of six other ORFs, with dispersed locations, was coordinated by act gene expression and they were named ecaA-ecaE. Their involvement in Act synthesis still needs to be demonstrated. Similarly, genes
whose expression was coordinated with red genes were identified and named ecr genes. Some of these were located distant from red locus genes. Interestingly, transcription of redD-independent and -dependent genes of the red locus was enhanced in an actII-ORF4 mutant.

Conclusions

The existence of the entire genome sequence and the ability to carry out global DNA
microarray analyses enabled study of the expression of functionally related genes
present within loci known to be involved in antibiotic synthesis in S. coelicolor, as well as the discovery of new genes that are distantly located but show coordinated
expression.

Reporter's comments

This study is a major contribution to our understanding of the gene regulation required
for antibiotic synthesis in S. coelicolor A3(2) and its results could only have been obtained by a global gene-expression analysis.
A major discovery are the sets of genes whose expression is coordinated with that
of genes known to be involved in Red or Act synthesis. This hints at possible involvement
of these new genes in the synthesis of Red or Act, or of other compounds whose production
might be controlled by similar mechanism(s). To pinpoint their precise biological
role(s) and to find possible applications, construction of mutants will be required
to perform further basic molecular genetic studies.

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